The invention relates to the coupling of thin-layer chromatography with mass spectrometry with ionization of the separated analyte substances by matrix-assisted laser desorption (MALDI).
The invention performs thin-layer chromatography (TLC) directly on a contactable, electrically conductive carrier plate which, after generation of small matrix crystals at the surface by suitable application of a matrix solution and drying, can be directly inserted into a mass spectrometer so that the chromatographically separated analyte substances contained in the matrix crystals, can be ionized and analyzed by MALDI mass spectrometry. The invention covers type and shape of the carrier plates and the application procedure of the matrix solution to the carrier plates for MALDI analysis.
While gas chromatography (GC) and liquid chromatography (HPLC) have been routinely coupled to mass spectrometers with substantial success for some time now and instruments for these xe2x80x9chyphenated techniquesxe2x80x9d (GC/MS, LC/MS) are commercially available from a number of vendors, that is not the case with thin-layer chromatography (TLC).
Thin-layer chromatography is used for many analytical tasks as a fast, simple procedure for performing quality inspections, determining reaction rates, progress of syntheses, separation of rare earth metals, doping tests in sport, in clinical chemistry and diagnostics, for narcotic tests in criminal proceedings, in forensic chemistry, residue analyses for pesticides, and many other fields. It is performed very. much like liquid chromatography with a liquid and a solid phase, whereby the migration of the liquid phase is solely caused by the capillary effect of the open-pore solid phase which is applied to a solid carrier plate in form of a thin layer. The procedure is simple and inexpensive. As with liquid chromatography, there are two types of thin layer chromatography: adsorption chromatography and distribution chromatography.
Knowledge of thin-layer chromatography is assumed here. Small volumes of various solutions containing the substance mixtures to be separated are applied as spots to the porous thin layer on a starting line. The edge of the carrier plates below the starting line is then immersed in a liquid mobile phase in a closed vessel at saturated vapor pressure for the liquid mobile phase, whereby the capillary migration of the mobile phase in the porous thin layer takes the individual substances with it. With different adsorption or distribution coefficients the analyte substances are transported at different rates and are thus separated. After removing and drying the carrier plates the analyte substances are distributed over the plate as spots along invisible tracks, which begin at the starting point of the mixtures and follow the front of the solvent. In this way ten to thirty mixtures can be separated on a single carrier plate simultaneously in parallel tracks.
For highly complex mixtures, two-dimensional thin-layer chromatography is also known. It consists of separating a mixture sample close to the edge in one direction at first. After drying the thin layer on the carrier plate a second separation of the substances, which have already been separated in one dimension, can be performed with a second mobile phase with different distribution coefficients, perpendicular to the first track.
Unless in the relatively rare case of colored substances, the substances distributed as spots cannot be seen with the naked eye. If the substances fluoresce, it is possible to see and measure the substances by exciting their fluorescence. In other cases the substances have to be stained after drying the plates. A fluorescing background can also be used for detection because the substances can be recognized by attenuation of fluorescence. However, under no circumstances xe2x88x92as in all chromatographic methods with merely intensity-indicating detectors - can unambiguous identification of the substances be performed with these methods of detection. Although there are indeed methods with high separation efficiency due to the development of perfectly uniform thin layers (so-called HPTLC=high performance thin layer chromatography), detection remains the weak point of thin-layer chromatography.
If one disregards the analysis of individual, usually scraped spots of substance by mass spectrometry, only relatively few extensive attempts have been made with mass spectrometric analysis of the substances separated by thin-layer chromatography. Probably the best and most recent work in this area is from J. T. Mehl, A. I. Gusev and D. M. Hercules xe2x80x9cCoupling Protocol for Thin Layer Chromatography/Matrix-assisted Laser Desorption Ionizationxe2x80x9d Chromatographia 46, 358 (1997). The authors report on various methods of mass spectrometric analysis in time-of-flight spectrometers, including their own experiments on direct MALDI ionization from the TLC plate, for which they report weak sensitivity, bad resolution, and rather bad mass resolving power. For this reason they developed an elaborate xe2x80x9csecond generationxe2x80x9d of coupling TLC/MS in order to avoid these disadvantages, and this consists of transferring the analyte substances from moistened TLC plate onto a MALDI sample support plate provided with a matrix, without any chromatographic thin layer. This more complicated method is presented by the authors as a much more satisfactory solution to the problem.
The inferior resolution of the former method must be due, in a manner as yet unexplained, to a lateral transport of the analyte substances during MALDI preparation. The inferior mass resolving power may have various causes, which can range from a charging of the thin layer to inferior definition of the ion-accelerating electric field by the base of the thin-layer plate.
Methods and devices should be found for simple and rapid qualitative or quantitative mass spectrometric analysis of analyte substances separated by thin-layer chromatography, with potential for automization and high sample throughput.
It is the basis of the invention to perform thin-layer chromatography directly on specially prepared and designed mass spectrometry sample support plates which are electrically conductive under the chromatographic thin layer and accessible to good electrical contact, and to extract the locally separated analyte substances from the porous chromatographic thin layer to the surface without any major lateral transport and generate a surface layer of matrix crystals containing the analyte substances on top of the porous chromatographic thin layer, introducing the carrier plates into the mass spectrometer, and qualitatively or quantitatively analyzing the analyte substances after ionization with matrix-assisted laser desorption (MALDI) by mass spectrometry directly from the thin-layer carrier plate.
By contrast with the description in the above-cited article, scans of the analyte substances can be obtained with a high level of sensitivity and reproducible concentration determination, with good substance separation and high mass resolving power. It seems to be crucial to bring the carrier plates in the mass spectrometer electrically up to acceleration potential without any major contact resistances. Furthermore, certain additional conditions in the extraction process for the analyte substances by the matrix solution have to be strictly maintained. The scans are surprisingly clean: generally they only contain the analyte ions and the ions of the matrix substance.
For determination by mass spectrometry it is possible to use both time-of-flight mass spectrometers and the various types of ion trap mass spectrometer (RF ion trap spectrometers or ion cyclotron resonance mass spectrometers).
Qualitative analysis, which means the identification of the substances, may be performed using libraries which contain both the masses and the mobile phase and layer specific Rf values of the substances (Rf=quotient of the migration rate of the substance and the migration rate of the mobile phase in the thin layer).
To improve identification of unknown substances, fragment ion spectra can be generated, for instance daughter ion spectra in ion trap spectrometers or the well-known PSD spectra in time-of-flight mass spectrometers (PSD=post source decay). These fragment spectra from MALDI ions are similar (with limitations) to the spectra which are obtained from the same substances by means of electron impact; for electron impact there are spectrum libraries containing hundreds of thousands of spectra. Studies of the differences in the fragmentation of protonated substances by contrast with electron impact fragmentation are being conducted at present.
Transport of the analyte substances from the bulk of the porous chromatographic layer into the surface layer of small matrix crystals, formed during the process, is critical. An adequately but not oversaturated soaking of the porous layer with a matrix solution followed by immediate drying is necessary for this. The drying process draws the matrix solution to the surface by capillary action, whereby the substances are entrained by being dissolved in accordance with their coefficient of distribution between porous layer and matrix solution. High solubility in the matrix solution helps to extract a large fraction each of the various substances. The entrainment displays surprisingly good quantitative reproducibility. The analyte substances are integrated into the scarcely visible matrix crystals forming at the surface or imbedded in grain boundaries. Lateral migration of the matrix solution in the thin layer can be avoided by the methods given below so that there is no measurable loss of lateral resolution of the thin-layer chromatogram or adulteration of the Rf values.
For soaking the porous layer with the matrix solution, printing has proved successful, for example with an elastic printing roller covered with the correct amount of solution or with an appropriate printing plate, as has spraying on a very fine mist of droplets. For the printing procedure, a printing plate can, for example, be used which is covered with a fine-pore foam rubber, whereby the foam rubber contains the matrix solution but is almost dry. Only when the printing plate is pressed on with relatively strong pressure is matrix solution given off to the highly absorbent thin layer, soaking the thin layer completely, and if the printing plate is lifted off carefully the excess of matrix solution is immediately also lifted off by the fine-pore foam rubber so that there can be no lateral migration. Printing is also best performed automatically in order to obtain a homogeneous printing layer of matrix solution.
For spraying it has been found that first the spraying procedure has to be completed within as short a time as possible, that second the droplets of the matrix solution have to be very small (approximately between one picoliter and one nanoliter in volume), but they must not dry out on their journey between the tip of the spray capillary and the carrier plate, and that third the density and duration of the mist of droplets must be such that continuous, saturated moisturizing or soaking of the porous thin-layer chromatography layer is achieved without any excess liquid. Spraying is therefore best performed automatically, whereby the matrix solution is best sprayed from below toward the carrier plate mounted on a carriage, which can be moved in two directions above the spray capillary.
The TLC/MALDI carrier plates used for the method should preferably be the same size as microtiter plates having useful surface areas of 78xc3x97108 millimeters because they can then be easily stacked and be picked up and processed by modern pipetting robots with their grab arms, and thus can also be transferred to the mass spectrometer. Specially designed carrier plates can also be mounted on frames so that the carrier plate and frame together produce the outer shape of a microtiter plate. Modern mass spectrometers process sample support plates the size of microtiter plates, whereby two-dimensional precision sensing of the surface is possible. The TLC/MALDI plates can be provided with a continuous thin layer, but also with individual thin-layer tracks, whereby it is favorable to provide the strips between the tracks with a hydrophobic surface.